1. Technical Field
The technical field of the present invention is generally the controlled nucleation of protein crystals, and more specifically non-photochemical laser induced nucleation combined with standard methods of protein crystal growth to result in a better quality of protein crystal.
2. Prior Art
A new method to induce and control nucleation discovered by Allan S. Myerson is known as non-photochemical laser induced nucleation. Myerson, A. S. et al., Phys. Rev. Lett. 77, 3475 (1996). In this method, short high-intensity laser pulses have been shown to induce nucleation in supersaturated solutions or urea.
The growth of protein crystals for structure determination relies on spontaneous nucleation. Seeded growth is normally not practical, since it would require a good quality protein crystal, which is not available. Other methods routinely used in small-molecule crystal growth such as cooling and the more common evaporation methods do not allow for nucleation control. Since the purpose of protein crystal growth experiments is to crystallize a few large high quality crystals, uncontrolled spontaneous nucleation is a significant problem. In the experiments using the xe2x80x9changing dropletxe2x80x9d technique for example, two to three nuclei per drop would usually result in large crystals of high quality while larger numbers of nuclei per drop would not. Nucleation is known to strongly depend on supersaturation. Rosenberger, F., Fundamentals of Crystal Growth (Springer-Verlag, Berlin, 1979); Chernov, A. A. , Modern Crystallography III Crystal Growth (Springer-Verlag, Berlin, 1984); and McPherson, A., Eur. J. Biochem., 189, 1 (1990). Such dependence demands precise regulation of protein supersaturation, precipitant concentration, pH, purity, thermal history and temperature.
Nucleation requires that protein molecules aggregate together in clusters and these clusters reach a critical size where they are thermodynamically favored to grow. In addition, the molecules must overcome the entropic barrier and arrange themselves in the appropriate lattice arrangement for the resulting nuclei to be crystalline. The higher the supersaturation, the smaller the critical radius. Rosenberger F. et al., J. Crystal Growth, 168, 1 (1996). Because the metastable zones of protein solutions are much wider than those of small molecules, the nucleation of crystalline proteins begins at very high levels of supersaturation (often several hundred to thousand percent). Pusey, M. L. , J. Crystal Growth, 110, 60 (1998); and McPherson, A., et al., Structure, 3, 759 (1995). Proteins often can nucleate and grow in an amorphous form. This generally is not desired. Recent work has shown that the second virial coefficient can be used to identify solution conditions favorable for crystallization. George, A. et al., Acta Cryst., D50, 361 (1994); Rosenbaum, D. F. et al., Phys. Rev. Lett., 76, 150 (1996); and Rosenbaum, D. F. et al., J. Crystal Growth, 169, 752 (1996).
In 1996, during a study designed to investigate whether supersaturated urea solutions would display non-linear optical properties similar to those of urea crystals because of the presence of ordered molecular clusters, the present inventor discovered serendipitously that the solutions nucleated. Myerson, A. S. et al., Phys. Rev. Lett. 77, 3475 (1996). The experiment involved the use of pulses of linearly polarized near infrared laser light. This wavelength of light was non-absorbing in urea solutions, which ruled out a photochemical mechanism. It was postulated that there was an alignment of molecules along the direction of the polarization due to the optical Kerr effect that reduced the entropy barrier to crystallization. Further studies of laser induced nucleation in the laboratories of the present inventor have demonstrated that the laser will induce nucleation in other substances (I-alanine, glycine, adipic acid, succinic acid), will reduce the nucleation induction time significantly when compared to an identical control, and will result in fewer crystals then observed in a spontaneously nucleated control. Matic, J., Masters Thesis, Polytechnic University.
Complete or highly detailed steric structures of proteins are indispensable information for an understanding of the specific properties and functions of the proteins. For example, information on the three-dimensional structure of a protein can serve as the basis for understanding the mechanism of function appearance in a biochemical system by an enzyme or hormone. In many fields, such as pharmaceutical science and chemical engineering, the three-dimensional structure of a protein can provide information for basic molecular design, specific drug design, protein engineering, biochemical synthesis and the like.
X-ray crystal structural analysis is the most cogent and high-accuracy means of obtaining three-dimensional steric structural information of proteins at atomic levels at present. Thus, to determine the three-dimensional structure of a protein by X-ray crystal structural analysis, one must have protein crystals of sufficient size and quality. Crystallization of a protein currently is performed by eliminating a solvent from an aqueous or anhydrous solution containing the protein, resulting in a supersaturated state and growing a crystal. However, there are several problems in protein crystallization conducted using the current art.
As discussed previously, it is difficult to obtain a crystal of excellent crystallinity or a large-sized single crystal. One reason may be that a biological macromolecule is readily influenced by gravity since its molecular weight is generally large and causes convection in the solution. Rosenberger, F., J. Cryst. Growth, 76, 618 (1986). This convection can reduce the crystal growth rate, or can cause anisotropic growth. Proteins also are sensitive to the crystallization conditions. The environment, pH, ionic strength and temperature of the solution, and type and dielectric constant of the buffer solution, and the like, can affect protein crystal growth. As a result, it has been difficult to obtain acceptable quantities of acceptable protein crystals, with most protein crystals being small, of less than excellent crystallinity, and in small quantities. Thus, crystallization of proteins is the weakest link in X-ray crystal structural analysis.
Others have used lasers to induce the crystallization of materials. For example, U.S. Pat. No. 4,330,363 to Biegesen et al. discloses thermal gradient control for enhanced laser-induced crystallization of predefined semiconductor areas and does not disclose or pertain to protein areas. Biegesen ""363 discloses a specific method of converting predefined areas of semiconductor material into single crystal areas and does not apply to the lased-induced nucleation of protein crystals or the controlled nucleation of protein crystals.
U.S. Pat. No. 4,737,232 to Flicstein et al. discloses a process for depositing and crystallizing a thin layer of organic material using laser energy. Flicstein ""232 discloses a specific method of depositing and crystallizing a thin layer of an organic material on a substrate, and using the laser to desorb material, and also does not apply to the lased-induced nucleation of protein crystals or the controlled nucleation of protein crystals.
U.S. Pat. No. 5,271,795 to Ataka et al. discloses a method of growing large crystals by locally controlling solution temperatures. Ataka ""795 discloses a method for growing protein crystals using the temperature dependence of solubility of a crystalline protein material, causing the protein crystals to be deposited by controlling the temperature of a localized portion of the solution. No laser is disclosed or suggested to induce or control the nucleation of protein crystals, the crystallization occurring by using warm water.
U.S. Pat. No. 5,683,935 to Miyamoto et al. discloses a method of growing semiconductor crystals only and does not disclose or pertain to protein areas. Miyamoto ""935 discloses a specific method of semiconductor crystallization by using laser light. This invention pertains to semiconductors, and does not have the same applicability to liquid solutions containing proteins.
U.S. Pat. No. 5,976,325 to Blanks discloses the laser-induced nucleation of purified aluminum hydrate crystals, including in supersaturated solutions. Although Blanks ""325 possibly can be applied to other supersaturated solution, there is no teaching or suggestion of using the process on organic materials or in fields unrelated to aluminums.
U.S. Pat. No. 6,055,106 to Grier et al. discloses a method and apparatus using laser light to assemble or direct particulate materials. Grier ""106 discloses a method for manipulating a plurality of biological objects including the crystallization of proteins. However, the invention is an optical trap that splits a single light beam into several, focuses the several light beams to form a focused spot for forming the optical trap, which is unrelated to the present invention.
Thus, it can be seen that no one has developed a successful method for the controlled nucleation of protein crystal growth that results in fewer larger protein crystals of better quality, a reduction in the nucleation induction time for growing protein crystals, and an increase in the overall rate of protein crystal growth. The present invention is directed to this end, namely, an improvement in the quality and size of protein crystals.
The present invention provides a method to control the nucleation of proteins in a liquid solution so that nucleation only occurs in a small part of the overall liquid solution. By controlling the number of nuclei, larger crystals result, which gives better results in x-ray structure analysis. One benefit of the invention is an improved rate of production and quality of protein crystals needed for determination of structures.
One purpose of controlling protein crystal growth is to produce protein crystals of superior quality and larger size for structure determination by x-ray crystallography. The quantity of protein crystals nucleated in a protein solution determines ultimate size, while solution composition, pH, supersaturation, temperature and purity control the protein crystal quality and structural resolution. It has been found that control of nucleation at appropriate crystallization conditions would improve the size and quality of protein crystal. In addition, it has been found that the ability to induce nucleation on demand (or to reduce the nucleation induction time) allows more successful protein crystal growth in shorter time periods.
The new method of the invention is a non-photochemical laser induced nucleation. Short high-intensity laser pulses are used to induce nucleation in supersaturated solutions including protein solutions. The laser induces nucleation only in the area where the beam is focused or passes through, resulting in much fewer nuclei than would be achieved by spontaneous nucleation. In addition, the laser reduces nucleation time significantly.
The method of non-photochemical laser induced nucleation of protein crystals as disclosed below, when combined with standard methods of protein crystal growth, results in fewer larger crystals of better quality. In addition, this method allows a reduction in the nucleation induction time so as to increase the overall rate of protein crystal growth. Thus, this results in an improvement in the quality and size of protein crystals and allows for more successful experiments per unit time.
Generally, the present invention provides a method for the controlled nucleation of protein crystal growth. The present invention further provides a method for the controlled nucleation of protein crystal growth that results in fewer larger protein crystals of better quality, a reduction in the nucleation induction time for growing protein crystals, and an improvement in the quality and size of protein crystals.
By employing the present invention, those skilled in the art can identify and optimize appropriate conditions of power, pulse length and polarization for the laser-induced nucleation of a number of different proteins so as to provide larger and higher diffraction quality protein crystals compared to current methods at identical conditions. Further, the present invention results in a reduction of the nucleation induction time needed for protein crystals when compared with spontaneous nucleation at identical conditions.
Further, this is the first time that laser-induced nucleation has been used to initiate the formation of protein crystals in protein solutions that will not spontaneously nucleate to form protein crystals.
These features and advantages of the present invention will become apparent to those of ordinary skill in the art when the following detailed description of the preferred embodiments is read in conjunction with the appended figure.